It is said that the third major challenge for a sustainable future (together with food security and energy) will be making the best use of limited supplies of pure water for both agricultural use and human consumption, and the remediation of marginal and contaminated water will be essential in achieving this. Already groundwater contamination, resulting from either natural geochemical processes or industrial activities such as mining, is a major problem in many countries.
Arsenic (As) is a groundwater contaminant that is ubiquitous in the environment and the two soluble forms, arsenite (AsIII) and arsenate (AsV), are toxic. Anthropogenic activity has resulted in widespread contamination of both soluble forms but AsIII is prevalent in anoxic environments, including most sources of drinking water. Countries affected include India, Bangladesh, Vietnam, USA, Germany, France, Hungary, Australia, Argentina, Mexico, Canada.
An important aspect of remediation is assessment and monitoring, and whilst laboratory methods exist that demonstrate high specificity and sensitivity (e.g. ICP-MS or HPLC) it is also possible, and indeed desirable, to measure analytes such as arsenite in the field using sensors. Ideally, the sensors should be low-cost, disposable and able to be readily adapted to multiple analytes that are commonly found together in contaminated water.
Many As field test kits are commercially available (e.g. from Industrial Systems, Inc, Hydrodyne) but these only detect total As, rather than the most toxic form, AsIII, which is dominant in anoxic drinking waters. Moreover, because As remediation preferentially removes AsV (e.g. by binding to iron hydroxide) and requires the pre-oxidation of AsIII, it is crucial to determine whether any AsIII remains in the water. The chemically based arsenic field kits rely on a colorimetric method which reduces the AsIII and AsV to the gas arsine which reacts with the mercuric bromide test strips. These kits require the training of personnel, are expensive (e.g. Arsenic, Quick II Hydrodyne kit US$4.24 per test and Ultra Low Quick II, Industrial Test Systems, Inc. US$6 per test) and have low sensitivity and reproducibility.
Whole cell biosensors have been developed for the detection of AsIII by a number of groups (e.g. Stocker et al. (2003) Environ. Sci. Technol. 37, 4743-4750). These methods are all based on colorimetric assays that sometimes require the use of a luminometer. They all use the regulatory mechanism of the Escherichia coli arsenic resistance system which detects both AsIII and antimonite (SbIII). The regulatory gene in this system, arsR, is fused to a reporter gene (e.g. luciferase gene, luxB) that when expressed after induction with AsIII produces a visible signal (e.g. fluorescence).
There are many problems with whole cell based AsIII biosensors, including: 1) the system is too complex and because of this has a slow response time (i.e. AsIII must enter cells followed by induction of regulator-reporter gene protein—this can take up to 24 hours for a response); 2) lack of specificity as the system does not discriminate between AsIII and SbIII; 3) incubation temperatures of 30° C. are often required; 4) colorimetric assay often requires use of a luminometer, which is not feasible at most field sites; and 5) use of genetically modified organisms always presents an additional problem. No whole cell biosensors for the detection of AsIII are commercially available.
A biosensor for AsIII has been developed using molybdenum-containing arsenite oxidase (known as “Aio” and also previously known as Aro and Aso; see Lett et al., Unified Nomenclature for Genes Involved in Prokaryotic Aerobic Arsenite Oxidation; J. Bacteriology, 4 Nov. 2011; p. 207-208) which is a member of the DMSO reductase family, prepared from chemolithoautotrophic Alphaproteobacterium Rhizobium sp. strain NT-26 (Santini et al. (2000) Appl. Environ. Microbiol. 66, 92-97).
AsIII oxidase catalyses the oxidation of AsIII to AsV and the suitability of this native enzyme for use as a biosensor has been tested and shown to detect down to 1 ppb AsIII, which is 10 times lower than the recommended WHO MCL (maximum contaminant level) of As in drinking water. Furthermore the native enzyme shows specificity for AsIII (Male et al. (2007) Anal. Chem. 79, 7831-7837). The biosensor comprises the enzyme directly linked to the surface of a multiwalled carbon nanotube-modified electrode, in which electron transfer proceeds directly from enzyme to electrode. The authors noted, however, that certain commonly-used electrode materials, in particular glassy carbon (GC), were not suitable for direct electron transfer in this configuration.
Heterologous expression of molybdenum-containing enzymes, especially members of the DMSO reductase family, is notoriously difficult. Recently, the dissimilatory arsenate reductase from Shewanella sp. str. ANA-3 was expressed in Escherichia coli but comparisons with the native enzyme were not made (Malasarn et al. (2008) J. Bacteriol. 190, 135-142). Expression was optimal when E. coli was grown anaerobically with DMSO although other electron acceptors for anaerobic growth were not tested and neither were different strains.
Since the entire native Aio is poorly expressed in a heterologous expression system, such as E. coli, use of this enzyme in routine detection of AsIII is not commercially viable.
As such, there is a need for improved methods and sensors for cheap and effective detection of AsIII in liquids such as drinking-water, waste-water and biological samples.